杜氏盐藻光系统Ⅱ反应中心蛋白D1基因的克隆及序列分析

Cloning and sequence analysis of photosystem Ⅱ reactor center proteins D1 gene of Dunaliella salina

目的:克隆杜氏盐藻光系统Ⅱ反应中心蛋白D1(psbA)基因cDNA片段。方法:根据莱茵衣藻、稻以及普通小球藻等真核生物psbA基因的氨基酸高度保守序列,设计一对简并引物,利用Trizol试剂提取杜氏盐藻细胞总RNA,通过RT-PCR获得杜氏盐藻psbA cDNA,PCR产物连接T载体转化大肠杆菌JM109,随机挑取数个菌落,筛选鉴定并测序,测序结果进行Blast比对分析和密码子偏爱性分析。结果:获得的cDNA片段长度为999 bp,编码333个氨基酸。其氨基酸序列与其他物种进行Blast比对,同源性分别为:衣藻89%、椭圆小球藻89%、莱茵衣藻89%、普通小球藻88%和玉米87%。psbA基因存在明显的密码子偏爱性,其(A+T)含量明显高于(G+C)含量。另外,所克隆的psbA序列编码赖氨酸残基的数量为1个。结论:所克隆的序列为杜氏盐藻psbA基因cDNA片段。

Aim：To clone cDNA fragment of photosystem Ⅱ reactor center proteins D1（psbA） gene from Dunaliella salina（D.salina）.Methods：One pair of degenerate primers was designed according to conserved motifs of the psbA of Chlamydomonas reinhardtii,Oryza sativa,and Chlorella vulgaris.And a total RNA of D.salina was extracted with Trizol reagent.A cDNA fragment from green algal D.salina was obtained through RT-PCR method.The PCR products were cloned into pMD18-T vector and then transformed into E.coli JM109.Several clones were selected randomly and screened to determine their sequences.In addition,homologous analysis of the deduced amino acid sequence was performed by blast and codon bias was investigated.Results： The obtained cDNA fragment was 999 base pairs in size,which encoded 333 amino acids.The sequence of psbA gene of D.salina shared high homologue with the following psbA： 89% of Chlamydomonas moewusii,89% of Chlorella ellipsoidea,89% of Chlamydomonas reinhardtii,88% of Chlorella vulgaris,and 87% of Zea mays,respectively.In addition,the analysis of codon bias showed that the codon usage of psbA gene of D.salina was apparently biased,and the content of（A＋T） was obviously higher than that of（G＋C）.And also,the number of Lys in the psbA gene was 1.Conclusion： The cloned sequence is psbA cDNA fragment of D.salina.